Publications by authors named "Shinichiro Motohashi"

87 Publications

[Application of iPS Cell-Derived NKT Cells to Cancer Immunotherapy].

Gan To Kagaku Ryoho 2020 Oct;47(10):1411-1414

Dept. of Medical Immunology, Graduate School of Medicine, Chiba University.

NKT cells are innate lymphocytes that express an invariant T cell receptor. Since activated NKT cells exert strong anti-tumor responses, NKT cells have been intensively studied for the purpose of their application to cancer immunotherapeutic approaches. Although human peripheral blood contained a very low fraction of NKT cells, and decreased number of NKT cells was also demonstrated in cancer-bearing patients, peripheral blood NKT cells can be activated by ligand-pulsed antigen presenting cells, and can produce a large amount of interferon-γ upon activation. The clinical trials of adoptive transfer of autologous NKT cells were already performed in patients with non-small cell lung cancer, and with head and neck cancer at Chiba University to show its effectiveness and limitation. Meanwhile, RIKEN reported NKT cell regeneration using iPS cell technology in mice, and subsequently established a protocol for regenerating NKT cells from human peripheral blood NKT cells using iPS cell technology. It was confirmed that the iPS cell-derived NKT cells (iPS-NKT) have sufficient expansion c apacity and potent direct and indirect cytotoxic activity in the humanized mice models, which suggests their therapeutic competence. We are currently planning an investigator-initiated clinical trial of allogeneic iPS-NKT cell therapy for head and neck cancer.
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October 2020

CD1d expression in glioblastoma is a promising target for NKT cell-based cancer immunotherapy.

Cancer Immunol Immunother 2020 Oct 31. Epub 2020 Oct 31.

Department of Medical Immunology, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba, 260-8670, Japan.

Glioblastoma is the most common and aggressive type of brain tumor with high recurrence and fatality rates. Although various therapeutic strategies have been explored, there is currently no effective treatment for glioblastoma. Recently, the number of immunotherapeutic strategies has been tested for malignant brain tumors. Invariant natural killer T (iNKT) cells play an important role in anti-tumor immunity. To address if iNKT cells can target glioblastoma to exert anti-tumor activity, we assessed the expression of CD1d, an antigen-presenting molecule for iNKT cells, on glioblastoma cells. Glioblastoma cells from 10 of 15 patients expressed CD1d, and CD1d-positive glioblastoma cells pulsed with glycolipid ligand induced iNKT cell-mediated cytotoxicity in vitro. Although CD1d expression was low on glioblastoma stem-like cells, retinoic acid, which is the most common differentiating agent, upregulated CD1d expression in these cells and induced iNKT cell-mediated cytotoxicity. Moreover, intracranial administration of human iNKT cells induced tumor regression of CD1d-positive glioblastoma in orthotopic xenografts in NOD/Shi-scid IL-2RγKO (NOG) mice. Thus, CD1d expression represents a novel target for NKT cell-based immunotherapy for glioblastoma patients.
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http://dx.doi.org/10.1007/s00262-020-02742-1DOI Listing
October 2020

Essential Role for CD30-Transglutaminase 2 Axis in Memory Th1 and Th17 Cell Generation.

Front Immunol 2020 21;11:1536. Epub 2020 Jul 21.

Department of Immunology, Graduate School of Medicine, Chiba University, Chiba, Japan.

Memory helper T (Th) cells are crucial for secondary immune responses against infectious microorganisms but also drive the pathogenesis of chronic inflammatory diseases. Therefore, it is of fundamental importance to understand how memory T cells are generated. However, the molecular mechanisms governing memory Th cell generation remain incompletely understood. Here, we identified CD30 as a molecule heterogeneously expressed on effector Th1 and Th17 cells, and CD30 effector Th1 and Th17 cells preferentially generated memory Th1 and Th17 cells. We found that CD30 mediated signal induced Transglutaminase-2 (TG2) expression, and that the TG2 expression in effector Th cells is essential for memory Th cell generation. In fact, -deficiency resulted in the impaired generation of memory Th1 and Th17 cells, which can be rescued by overexpression of TG2. Furthermore, ()-deficient CD4 T cells failed to become memory Th cells. As a result, T cells from -deficient mice displayed impaired antigen-specific antibody production and attenuated Th17-mediated allergic responses. Our data indicate that CD30-induced TG2 expression in effector Th cells is essential for the generation of memory Th1 and Th17 cells, and that CD30 can be a marker for precursors of memory Th1 and Th17 cells.
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http://dx.doi.org/10.3389/fimmu.2020.01536DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7385138PMC
April 2021

A phase I study of loco-regional immunotherapy by transbronchial injection of α-galactosylceramide-pulsed antigen presenting cells in patients with lung cancer.

Clin Immunol 2020 06 6;215:108457. Epub 2020 May 6.

Department of Medical Immunology, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan. Electronic address:

We conducted a phase I study of the trans-bronchial injection of α-galactosylceramide (αGalCer)-pulsed antigen presenting cells (APCs) to evaluate their safety, immune responses, and anti-tumor activities. Patients with advanced or recurrent non-small cell lung cancer (NSCLC) refractory to standard treatments were eligible. αGalCer-pulsed APCs were administered intratumorally or intranodally by bronchoscopy. Twenty-one patients were enrolled in this study. No severe adverse events related to the cell therapy were observed during this study in any patient. After αGalCer-pulsed APCs were administrated, increased iNKT cell numbers were observed in PBMCs from eight cases, and IFN-γ producing cells were increased in the peripheral blood of 10 cases. Regarding clinical responses, one case exhibited a partial response and eight were classified as stable disease. In the tumor microenvironment, IFN-γ expression was upregulated after treatment in partial response or stable disease cases and TGF-β was upregulated in progressive disease cases.
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http://dx.doi.org/10.1016/j.clim.2020.108457DOI Listing
June 2020

Differential gene analysis during the development of obliterative bronchiolitis in a murine orthotopic lung transplantation model: A comprehensive transcriptome-based analysis.

PLoS One 2020 8;15(5):e0232884. Epub 2020 May 8.

Department of General Thoracic Surgery, Chiba University Graduate School of Medicine, Chiba, Japan.

Background: Obliterative bronchiolitis (OB) is a known issue during minor histocompatibility antigen (mHA) disparity during lung transplantation. This study evaluated gene expression in a murine orthotropic lung transplantation model using microarray analysis.

Methods: Left lungs from C57BL/10(H-2b) donor mice were transplanted into mHA-mismatched C57BL/6(H-2b) recipient mice. Three groups (OB, non-OB, and sham controls) were confirmed pathologically and analyzed. Gene expression changes in the lung grafts were determined by microarray and immunohistochemical staining, and genes were verified by quantitative PCR in the lungs and mediastinal lymph nodes (LNs).

Results: A total of 1343 genes were upregulated in the OB lungs compared to the sham group. Significant upregulation was observed for genes related to innate, e.g. Tlr2 and CCL3 and adaptive immunity, e.g. H2-ab1 and Il-21. Positive labeling for MHC class II antigen was observed in the bronchial epithelium of OB accompanied with B cells. We found increased Tlr2, Ccl3, H2-ab1, Il-21, Ighg3, Ifng, and Pdcd1 mRNA expression in the OB lung, and increased Il-21, Ighg3, and Pdcd1 expression in the OB LNs.

Conclusions: Adaptive and innate immune reactions were involved in OB after lung transplantation, and genetic examination of related genes could be used for detection of OB.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0232884PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7209239PMC
August 2020

Activated invariant natural killer T cells directly recognize leukemia cells in a CD1d-independent manner.

Cancer Sci 2020 Jul 19;111(7):2223-2233. Epub 2020 Jun 19.

Department of Medical Immunology, Graduate School of Medicine, Chiba University, Chiba, Japan.

Invariant natural killer T (iNKT) cells are innate-like CD1d-restricted T cells that express the invariant T cell receptor (TCR) composed of Vα24 and Vβ11 in humans. iNKT cells specifically recognize glycolipid antigens such as α-galactosylceramide (αGalCer) presented by CD1d. iNKT cells show direct cytotoxicity toward CD1d-positive tumor cells, especially when CD1d presents glycolipid antigens. However, iNKT cell recognition of CD1d-negative tumor cells is unknown, and direct cytotoxicity of iNKT cells toward CD1d-negative tumor cells remains controversial. Here, we demonstrate that activated iNKT cells recognize leukemia cells in a CD1d-independent manner, however still in a TCR-mediated way. iNKT cells degranulated and released Th1 cytokines toward CD1d-negative leukemia cells (K562, HL-60, REH) as well as αGalCer-loaded CD1d-positive Jurkat cells. The CD1d-independent cytotoxicity was enhanced by natural killer cell-activating receptors such as NKG2D, 2B4, DNAM-1, LFA-1 and CD2, but iNKT cells did not depend on these receptors for the recognition of CD1d-negative leukemia cells. In contrast, TCR was essential for CD1d-independent recognition and cytotoxicity. iNKT cells degranulated toward patient-derived leukemia cells independently of CD1d expression. iNKT cells targeted myeloid malignancies more than acute lymphoblastic leukemia. These findings reveal a novel anti-tumor mechanism of iNKT cells in targeting CD1d-negative tumor cells and indicate the potential of iNKT cells for clinical application to treat leukemia independently of CD1d.
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http://dx.doi.org/10.1111/cas.14428DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7385353PMC
July 2020

Phase II study of α-galactosylceramide-pulsed antigen-presenting cells in patients with advanced or recurrent non-small cell lung cancer.

J Immunother Cancer 2020 03;8(1)

Department of Medical Immnunology, Graduate School of Medicine, Chiba University, Chiba, Japan

Background: Invariant natural killer T (iNKT) cells produce copious amounts of cytokines in response to specific glycolipid antigens such as α-galactosylceramide (αGalCer) presented by CD1d-expressing antigen-presenting cells (APCs), thus orchestrating other immune cells to fight tumors. Because of their ability to induce strong antitumor responses activated by αGalCer, iNKT cells have been studied for their application in cancer immunotherapy. In our previous phase I/II trial in non-small cell lung cancer (NSCLC) patients who had completed the standard treatment, we showed a relatively long median survival time without severe treatment-related adverse events. Based on these results, we performed a phase II trial to evaluate clinical responses, safety profiles and immune responses as a second-line treatment for advanced NSCLC.

Methods: Patients with advanced or recurrent NSCLC refractory to first-line chemotherapy were eligible. αGalCer-pulsed APCs were intravenously administered four times. Overall survival time was evaluated as the primary endpoint. The safety profile and immune responses after APC injection were also monitored. This study was an open label, single-arm, phase II clinical trial performed at Chiba University Hospital, Japan.

Results: Thirty-five patients were enrolled in this study, of which 32 (91.4%) completed the trial. No severe adverse events related to the treatment were observed. The estimated median survival time of the 35 cases was 21.9 months (95% CI, 14.8 to 26.0). One case (2.9%) showed a partial response, 14 cases (40.0%) remained as stable disease, and 19 cases (54.3%) were evaluated as progressive disease. The geometric mean number of iNKT cells in all cases was significantly decreased and the mean numbers of natural killer (NK) cells, interferon-γ-producing cells in response to αGalCer, and effector CD8+ T cells were significantly increased after the administration of αGalCer-pulsed APCs.

Conclusions: The intravenous administration of αGalCer-pulsed APCs was well-tolerated and was accompanied by prolonged overall survival. These results are encouraging and warrant further evaluation in a randomized phase III trial to demonstrate the survival benefit of this immunotherapy.

Trial Registration Number: UMIN000007321.
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http://dx.doi.org/10.1136/jitc-2019-000316DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7078938PMC
March 2020

Immunological features of a lung cancer patient achieving an objective response with anti-programmed death-1 blockade therapy.

Cancer Sci 2020 Jan 29;111(1):288-296. Epub 2019 Nov 29.

Department of Medical Immunology, Graduate School of Medicine, Chiba University, Chiba, Japan.

The role of immune checkpoint inhibitors in metastatic lung cancer has been established in recent years and the pretherapeutic profiles of the tumor microenvironment in responders have been increasingly reported. The role of salvage surgery and the immune profiles of the posttherapeutic specimens in patients achieving an objective response have rarely been studied. We report a case of metastatic lung cancer treated by anti-programmed death-1 Ab followed by surgical resection. The immune status of the tumor was assessed, showing germinal center formation, memory B cell infiltration, and a high frequency of interferon gamma -secreting T cells.
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http://dx.doi.org/10.1111/cas.14222DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6942425PMC
January 2020

Regulatory T cells induce CD4 NKT cell anergy and suppress NKT cell cytotoxic function.

Cancer Immunol Immunother 2019 Dec 22;68(12):1935-1947. Epub 2019 Oct 22.

Department of Otorhinolaryngology, Head and Neck Surgery, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba, 260-8670, Japan.

Background: Due to the strong tumoricidal activities of activated natural killer T (NKT) cells, invariant NKT cell-based immunotherapy has shown promising clinical efficacy. However, suppressive factors, such as regulatory T cells (Tregs), may be obstacles in the use of NKT cell-based cancer immunotherapy for advanced cancer patients. Here, we investigated the suppressive effects of Tregs on NKT cells and the underlying mechanisms with the aim to improve the antitumor activities of NKT cells.

Methods: Peripheral blood samples were obtained from healthy donors, patients with benign tumors, and patients with head and neck squamous cell carcinoma (HNSCC). NKT cells, induced with α-galactosylceramide (α-GalCer), and monocyte-derived dendritic cells (DCs) were co-cultured with naïve CD4 T cell-derived Tregs to investigate the mechanism of the Treg suppressive effect on NKT cell cytotoxic function. The functions and phenotypes of NKT cells were evaluated with flow cytometry and cytometric bead array.

Results: Treg suppression on NKT cell function required cell-to-cell contact and was mediated via impaired DC maturation. NKT cells cultured under Treg-enriched conditions showed a decrease in CD4 NKT cell frequency, which exert strong tumoricidal responsiveness upon α-GalCer stimulation. The same results were observed in HNSCC patients with significantly increased effector Tregs.

Conclusion: Tregs exert suppressive effects on NKT cell tumoricidal function by inducing more CD4 NKT cell anergy and less CD4 NKT cell anergy. Both Treg depletion and NKT cell recovery from the anergy state may be important for improving the clinical efficacy of NKT cell-based immunotherapy in patients with advanced cancers.
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http://dx.doi.org/10.1007/s00262-019-02417-6DOI Listing
December 2019

Soluble factors derived from neuroblastoma cell lines suppress dendritic cell differentiation and activation.

Cancer Sci 2019 Mar 1;110(3):888-902. Epub 2019 Feb 1.

Department of Medical Immunology, Graduate School of Medicine, Chiba University, Chiba, Japan.

Dendritic cells (DC) play a key role in the initiation of both antitumor immunity and immunological tolerance. It has been demonstrated that exposure to soluble factors produced by tumor cells modulates DC functions and induces tolerogenic DC differentiation. In this study, we investigated the effects of neuroblastoma cell line-derived soluble factors on DC differentiation. Monocytes isolated from healthy volunteers were incubated with interleukin (IL)-4 and granulocyte-macrophage colony-stimulating factor in the presence of culture supernatants from neuroblastoma cell lines. The culture supernatants from neuroblastoma cell lines, such as NLF and GOTO, partially blocked both downregulation of CD14 and upregulation of CD1a, and dramatically decreased IL-12 and tumor necrosis factor (TNF)-α production from mature DC, while no effect of SH-SY5Y cell supernatant was noted. In addition, IL-6 and IL-10 production from monocytes was increased by the supernatants of NLF and GOTO cells at 24 hours after incubation. Furthermore, we evaluated DC functions through stimulation of invariant natural killer T (iNKT) cells. α-Galactosylceramide-pulsed DC co-cultured with supernatants of NLF cells were unable to sufficiently stimulate iNKT cells. The decreased ability of iNKT cells to produce interferon (IFN)-γ after stimulation with neuroblastoma cell line supernatant-cultured DC was reversed by addition of IL-12. CD40 expression and IL-12 production in NLF-sup-treated DC were increased by addition of exogenous IFN-γ. These results indicate that tolerogenic DC are induced in the neuroblastoma tumor microenvironment and attenuate the antitumor effects of iNKT cells. Interactions between iNKT cells and αGalCer-pulsed DC have the potential to restore the immunosuppression of tolerogenic DC through IFN-γ production.
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http://dx.doi.org/10.1111/cas.13933DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6398884PMC
March 2019

Activated iNKT cells enhance the anti-tumor effect of antigen specific CD8 T cells on mesothelin-expressing salivary gland cancer.

BMC Cancer 2018 Dec 17;18(1):1254. Epub 2018 Dec 17.

Department of Otorhinolaryngology, Head and Neck Surgery, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba, 260-8670, Japan.

Background: Salivary gland cancers are not sensitive to conventional radiotherapy or chemotherapy regimens. Therefore, the development of a new treatment strategy is of critical importance for improving the prognosis. We examined the expression of mesothelin molecules in salivary gland cancers and the efficacy of adoptive cell therapy based on mesothelin-specific chimeric antigen receptor transduced T cells.

Methods: The expression of mesothelin molecule was studied in salivary gland cancer samples obtained from 16 patients as well as a salivary gland cancer cell line (A-253) and five other cell lines. The activation of mesothelin-specific chimeric antigen receptor-expressing CD8 T cells after stimulation with mesothelin and the effects of invariant natural killer T cells on this activation were evaluated.

Results: Mesothelin was detected in the A-253 cells and the surgical specimens except for the case of squamous cell carcinoma to various degrees. Following stimulation with mesothelin expressing cancer cells, chimeric antigen receptor T cells were dose-dependently activated; this activation was enhanced by co-culture with invariant natural killer T cells and subsequently abrogated by treatment with anti-interferon-γ antibodies. Furthermore, the cytotoxicity of chimeric antigen receptor T cells against various cancer cells was further augmented by invariant natural killer T cells.

Conclusions: The use of adoptive transfer with mesothelin-specific chimeric antigen receptor-expressing CD8 T cells against salivary gland cancers is an effective therapy and invariant natural killer T cells are expected to be used in adjuvant treatment for T cell-based immunotherapy.
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http://dx.doi.org/10.1186/s12885-018-5179-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6296067PMC
December 2018

Role of leukotriene B4 12-hydroxydehydrogenase in α-galactosylceramide-pulsed dendritic cell therapy for non-small cell lung cancer.

Biochem Biophys Res Commun 2018 11 15;506(1):27-32. Epub 2018 Oct 15.

Department of Medical Immunology, Graduate School of Medicine, Chiba University, 2608670, Chiba, Japan. Electronic address:

Invariant natural killer T (iNKT) cells exhibit potent antitumor effects upon activation by recognizing a specific glycolipid antigen. We previously performed phase I-II clinical studies to utilize iNKT cells using α-galactosylceramide-pulsed dendritic cells and identified leukotriene B4 12-hydroxydehydrogenase (LTB4DH) as a biomarker highly expressed in T cells derived from non-small cell lung cancer (NSCLC) patients who showed prolonged survival in respond to the iNKT cell immunotherapy. Because LTB4DH expression correlated with prolonged survival of NSCLC patients, we considered LTB4DH to play a role in iNKT cell immunotherapy. We herein demonstrate that the overexpression of LTB4DH in CD4 or CD8 T cells increases interferon-γ production and tumoricidal activity in the presence of prostaglandin E. Moreover, the expression of granzyme a, granzyme b, and perforin mRNA was increased in LTB4DH-overexpressing cells.
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http://dx.doi.org/10.1016/j.bbrc.2018.10.048DOI Listing
November 2018

TGF-β suppresses RasGRP1 expression and supports regulatory T cell resistance against p53-induced CD28-dependent T-cell apoptosis.

Eur J Immunol 2018 12 24;48(12):1938-1943. Epub 2018 Oct 24.

Department of Microbiology and Immunology, Loyola University, Chicago, IL, USA.

Thymus-derived regulatory T cells (tTregs) play pivotal roles in immunological self-tolerance and homeostasis. A majority of tTregs are reactive to self-antigens and are constantly exposed to antigenic stimulation. Despite this continuous stimulation, tTreg and conventional T-cell populations remain balanced during homeostasis, but the mechanisms controlling this balance are unknown. We previously reported a form of activation-induced cell death, which is dependent on p53 (p53-induced CD28-dependent T-cell apoptosis, PICA). Under PICA-inducing conditions, tTregs survive while a majority of conventional T cells undergo apoptosis, suggesting there is a survival mechanism that protects tTregs. Here, we report that the expression of RasGRP1 (Ras guanyl-releasing protein 1) is required for PICA, as conventional T cells isolated from RasGRP1-deficient mice become resistant to PICA. After continuous stimulation, tTregs express a substantially lower amount of RasGRP1 compared to conventional T cells. This reduced expression of RasGRP1 is dependent on TGF-β, as addition of TGF-β to conventional T cells reduces RasGRP1 expression. Conversely, RasGRP1 expression in tTregs increases when TGF-β signaling is inhibited. Together, these data show that RasGRP1 expression is repressed in tTregs by TGF-β signaling and suggests that reduced RasGRP1 expression is critical for tTregs to resist apoptosis caused by continuous antigen exposure.
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http://dx.doi.org/10.1002/eji.201847587DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6368088PMC
December 2018

Clinical Application of iNKT Cell-mediated Anti-tumor Activity Against Lung Cancer and Head and Neck Cancer.

Front Immunol 2018 7;9:2021. Epub 2018 Sep 7.

Department of Medical Immunology, Graduate School of Medicine, Chiba University, Chiba, Japan.

Invariant natural killer T (iNKT) cells produce copious amounts of cytokines in response to T-cell receptor (TCR) stimulation by recognizing antigens such as α-galactosylceramide (α-GalCer) presented on CD1d; thus, orchestrating other immune cells to fight against pathogen infection and tumors. Because of their ability to induce strong anti-tumor responses and the convenience of their invariant TCR activated by a synthetic ligand, α-GalCer, iNKT cells have been intensively studied for application in immunotherapeutic approaches to treat cancer patients in the clinic. Here, we summarize the clinical trials of iNKT cell based immunotherapy for non-small cell lung cancer, and head and neck cancer. Although solid tumors are thought to be refractory to immunotherapeutic approaches, our clinical trials showed that the intravenous injection of α-GalCer-pulsed antigen presenting cells (APCs) activated endogenous iNKT cells and iNKT cell dependent responses. Moreover, an increase in the number of IFN-γ producing cells in PBMCs was associated with prolonged survival. The marked infiltration of iNKT cells and the accumulation of conventional T cells in the tumor microenvironment were also observed after the administration of α-GalCer-pulsed APCs and/or activated iNKT cells. In cases of advanced head and neck squamous cell carcinoma, the increased accumulation of iNKT cells in the tumor microenvironment was correlated with objective clinical responses. We will also discuss potential combination therapies of iNKT cell based immunotherapy to achieve enhanced anti-tumor activity and provide better treatment options for these patients.
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http://dx.doi.org/10.3389/fimmu.2018.02021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6137091PMC
October 2019

CD69 prevents PLZF innate precursors from prematurely exiting the thymus and aborting NKT2 cell differentiation.

Nat Commun 2018 09 14;9(1):3749. Epub 2018 Sep 14.

Department of Immunology, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba, 260-8670, Japan.

While CD69 may regulate thymocyte egress by inhibiting S1P expression, CD69 expression is not thought to be required for normal thymocyte development. Here we show that CD69 is in fact specifically required for the differentiation of mature NKT2 cells, which do not themselves express CD69. Mechanistically, CD69 expression is required on CD24 PLZF innate precursors for their retention in the thymus and completion of their differentiation into mature NKT2 cells. By contrast, CD69-deficient CD24 PLZF innate precursors express S1P and prematurely exit the thymus, while S1P inhibitor treatment of CD69-deficient mice retains CD24 PLZF innate precursors in the thymus and restores NKT2 cell differentiation. Thus, CD69 prevents S1P expression on CD24 PLZF innate precursor cells from aborting NKT2 differentiation in the thymus. This study reveals the importance of CD69 to prolong the thymic residency time of developing immature precursors for proper differentiation of a T cell subset.
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http://dx.doi.org/10.1038/s41467-018-06283-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6138739PMC
September 2018

Crucial role of CD69 in anti-tumor immunity through regulating the exhaustion of tumor-infiltrating T cells.

Int Immunol 2018 11;30(12):559-567

Department of Immunology, Graduate School of Medicine, Chiba University, Chuo-ku, Chiba, Japan.

The introduction of immune checkpoint inhibitors in cancer treatment highlights the negative regulation of anti-tumor immunity, such as effector T-cell exhaustion in the tumor microenvironment. However, the mechanisms underlying the induction and prevention of T-cell exhaustion remain largely unknown. We found that CD69, a type II glycoprotein known to regulate inflammation through T-cell migration and retention in tissues, plays an important role in inducing the exhaustion of tumor-infiltrating T cells. Cd69-/- mice showed reduced tumor growth and metastasis in a 4T1-luc2 murine breast cancer model, in which increased numbers of tumor-infiltrating lymphocytes, relatively little T-cell exhaustion, and enhanced IFNγ production were observed. Anti-CD69 monoclonal antibody treatment attenuated the T-cell exhaustion and tumor progression in tumor-bearing mice. These findings highlight a novel role of CD69 in controlling the tumor immune escape mediated by T-cell exhaustion and indicate that CD69 is a novel target for cancer immunotherapy.
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http://dx.doi.org/10.1093/intimm/dxy050DOI Listing
November 2018

Immunosuppressive property of submandibular lymph nodes in patients with head and neck tumors: differential distribution of regulatory T cells.

BMC Res Notes 2018 Jul 16;11(1):479. Epub 2018 Jul 16.

Department of Otorhinolaryngology, Head and Neck Surgery, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba, 260-8670, Japan.

Objective: Different sensitizations and immune responses are thought to be induced in response to antigens at different mucosal sites between the oral floor and nose. The aim of this study was to investigate differences in the distributions of lymphocyte subsets in the submandibular (SM) and upper jugular (UJ) lymph nodes (LNs), which are supposed to be regional LNs of the oral floor and nasal mucosa, respectively. SMLNs and UJLNs were collected from patients with head and neck tumors who underwent surgical resection. The populations of T cells, Natural Killer (NK) cells, Natural Killer T (NKT) cells, regulatory T cells (Tregs) and dendritic cells (DCs) in LNs without metastasis were analyzed by flow cytometry. The high-affinity IgE receptor (FcεRI) expression of LN cells were also evaluated.

Results: The proportions of CD4CD25Foxp3 Tregs, CD4CD45RAFoxp3 effector Tregs and FcεRIαCD33CD11c DCs were significantly larger in SMLNs compared with UJLNs, while those of CD3 T cells, CD3CD56 NK cells, CD3Vα24Vβ11 NKT cells, and CD123CD303 DCs did not show any significant differences between SMLNs and UJLNs. The differential distributions of CD4CD25Foxp3 Tregs were observed regardless of tumor region, LN metastasis and clinical staging. These data indicate that SMLNs may have immunosuppressive properties compared with UJLNs.
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http://dx.doi.org/10.1186/s13104-018-3587-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6048882PMC
July 2018

Double-Stranded RNA Derived from Lactic Acid Bacteria Augments Th1 Immunity Interferon-β from Human Dendritic Cells.

Front Immunol 2018 23;9:27. Epub 2018 Jan 23.

Biomedical Research Institute, National Institute for Advanced Industrial Science and Technology (AIST), Tsukuba, Japan.

Lactic acid bacteria (LAB) are one of the major commensal species in the small intestine and known for contributing to maintenance of protective immunity and immune homeostasis. However, currently there has been no evidence regarding the cellular mechanisms involved in the probiotic effects of LAB on human immune cells. Here, we demonstrated that LAB double-stranded RNA (dsRNA) triggered interferon-β (IFN-β) production by human dendritic cells (DCs), which activated IFN-γ-producing T cells. Interleukin-12 (IL-12) secretion from human DCs in response to LAB was abrogated by depletion of bacterial dsRNA, and was attenuated by neutralizing IFN-β, indicating LAB dsRNA primarily activated the IFN-β/IL-12 pathway. Moreover, the induction of IL-12 secretion from DCs by LAB was abolished by the inhibition of endosomal acidification, confirming the critical role of the endosomal digestion of LAB. In a coculture of human naïve CD4 T cells and BDCA1 DCs, DCs stimulated with LAB containing dsRNA induced IFN-γ-producing T cells. These results indicate that human DCs activated by LAB enhance Th1 immunity depending on IFN-β secretion in response to bacterial dsRNA.
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http://dx.doi.org/10.3389/fimmu.2018.00027DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5787129PMC
February 2019

Invariant natural killer T infiltration in neuroblastoma with favorable outcome.

Pediatr Surg Int 2018 Feb 10;34(2):195-201. Epub 2017 Oct 10.

Department of Medical Immunology, Chiba University Graduate School of Medicine, Chiba, Japan.

Background: Tumor immunity has been suggested to play a key role in clinical and biological behavior of neuroblastomas. Given that CD1-restricted invariant natural killer T (iNKT) cells enhance both innate and acquired tumor immunity, we investigated the expression of the iNKT-cell-specific T-cell receptor Vα24-Jα18 in neuroblastoma tissues and its correlation with clinical and biological characteristics.

Methods: Using real- time quantitative PCR, we quantified the expression of Vα24-Jα18 in untreated tumor samples from 107 neuroblastoma cases followed in our institution and analyzed the correlation between the presence of infiltrated iNKT cells and clinical characteristics or patients' outcome.

Results: Vα24-Jα18 receptor was detected in 62 untreated cases (57.9%). The expression was significantly higher in stages 1, 2, 3, or 4S (P = 0.0099), in tumors with low or intermediate risk (P = 0.0050), with high TrkA expression (P = 0.0229), with favorable histology (P = 0.0026), with aneuploidy (P = 0.0348), and in younger patients (P = 0.0036). The overall survival rate was significantly higher in patients with iNKT-cell infiltration (log-rank; P = 0.0089).

Conclusions: Since tumor-infiltrating iNKT cells were predominantly observed in neuroblastomas undergoing spontaneous differentiation and/or regression, we suggest that iNKT cells might play a key role in these processes.
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http://dx.doi.org/10.1007/s00383-017-4189-xDOI Listing
February 2018

Frequency and proliferative response of circulating invariant natural killer T cells in pediatric patients with malignant solid tumors.

Pediatr Surg Int 2018 Feb 10;34(2):169-176. Epub 2017 Oct 10.

Department of Medical Immunology, Graduate School of Medicine, Chiba University, Chiba, Japan.

Background: Invariant natural killer T (iNKT) cells play an important role in tumor immunity, enhancing both innate and acquired immunity. We have previously shown the enhancement of antibody-dependent cellular cytotoxicity against neuroblastoma by activated iNKT cells. As a first step towards clinical application, we studied the frequency and proliferative response of circulating iNKT cells in children with and without cancer.

Methods: Blood samples were collected from 10 patients with pediatric malignant solid tumors and 11 patients with non-neoplastic diseases (control). The frequency of circulating iNKT cells was quantified by flow cytometry. Whole peripheral blood mononuclear cells were then stimulated with α-galactosylceramide (α-GalCer) for 7 days, and the expansion rate of the iNKT-cell fraction was assessed.

Results: The frequency of iNKT cells in the patients of the cancer and control group did not differ to a statistically significant extent. The iNKT-cell population increased after α-GalCer stimulation in all cases. The iNKT cells of patients who had undergone intensive chemotherapy also had the potential to expand in vitro.

Conclusions: Unlike adult cancer patients, the numbers of circulating iNKT cells were not decreased in pediatric cancer patients. α-GalCer stimulation induced a proliferative response in all of the patients.
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http://dx.doi.org/10.1007/s00383-017-4185-1DOI Listing
February 2018

[II. Overview and Future Outlook of Immune Cell Therapy for Lung Cancer].

Gan To Kagaku Ryoho 2017 Aug;44(8):650-654

Dept. of Medical Immunology, Graduate School of Medicine, Chiba University.

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August 2017

Myosin light chains 9 and 12 are functional ligands for CD69 that regulate airway inflammation.

Sci Immunol 2016 Sep 16;1(3):eaaf9154. Epub 2016 Sep 16.

Department of Immunology, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan.

Recent decades have witnessed a rapid worldwide increase in chronic inflammatory disorders such as asthma. CD4 T helper 2 cells play critical roles in the pathogenesis of allergic airway inflammation, and CD69 expression on activated CD4 T cells is required to induce allergic inflammation in tissues. However, how CD69 mechanistically controls allergic inflammation remains poorly defined. In lymphoid tissues, CD69 regulates cellular retention through inhibition of S1P1 expression and requires no specific ligands to function. In contrast, we show herein that myosin light chain (Myl) 9 and Myl12 are new functional ligands for CD69. Blockade of CD69-Myl9/12 interaction ameliorates allergic airway inflammation in ovalbumin-induced and house dust mite-induced mouse models of asthma. Within the inflamed mouse airways, we found that the expression of Myl9/12 was increased and that platelet-derived Myl9/12 localized to the luminal surface of blood vessels and formed intravascular net-like structures. Analysis of nasal polyps of eosinophilic chronic rhinosinusitis patients revealed that Myl9/12 expression was increased in inflammatory lesions and was distributed within net-like structures in the intravascular space. In addition, we detected Myl9/12 in perivascular spaces where many CD69 cells were positioned within Myl9/12 structures. Thus, CD69-Myl9/12 interaction is a key event in the recruitment of activated CD69 T cells to inflamed tissues and could be a therapeutic target for intractable airway inflammatory diseases.
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http://dx.doi.org/10.1126/sciimmunol.aaf9154DOI Listing
September 2016

Crucial role for CD69 in allergic inflammatory responses: CD69-Myl9 system in the pathogenesis of airway inflammation.

Immunol Rev 2017 07;278(1):87-100

Department of Immunology, Graduate School of Medicine, Chiba University, Chiba, Japan.

CD69 has been known as an early activation marker of lymphocytes; whereas, recent studies demonstrate that CD69 also has critical functions in immune responses. Early studies using human samples revealed the involvement of CD69 in various inflammatory diseases including asthma. Moreover, murine disease models using Cd69 mice and/or anti-CD69 antibody (Ab) treatment have revealed crucial roles for CD69 in inflammatory responses. However, it had not been clear how the CD69 molecule contributes to the pathogenesis of inflammatory diseases. We recently elucidated a novel mechanism, in which the interaction between CD69 and its ligands, myosin light chain 9, 12a and 12b (Myl9/12) play a critical role in the recruitment of activated T cells into the inflammatory lung. In this review, we first summarize CD69 function based on its structure and then introduce the evidence for the involvement of CD69 in human diseases and murine disease models. Then, we will describe how we discovered CD69 ligands, Myl9 and Myl12, and how the CD69-Myl9 system regulates airway inflammation. Finally, we will discuss possible therapeutic usages of the blocking Ab to the CD69-Myl9 system.
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http://dx.doi.org/10.1111/imr.12559DOI Listing
July 2017

CD45RAFoxp3 regulatory T cells have a negative impact on the clinical outcome of head and neck squamous cell carcinoma.

Cancer Immunol Immunother 2017 Oct 27;66(10):1275-1285. Epub 2017 May 27.

Department of Otorhinolaryngology, Head and Neck Surgery, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba, 260-8670, Japan.

Background: Although regulatory T cells (Tregs) are thought to play an important role in immune suppression, their clinical significance in head and neck squamous cell carcinoma (HNSCC) is unclear. A recent study reported Tregs could be divided into functional subsets based on the expression of CD45RA and Foxp3.

Method: The frequency of circulating Treg subsets was analyzed in patients with HNSCC and compared with the frequency in patients with benign tumors. The association of Treg subsets with the frequency of lymphocyte subsets, status of progression, clinical course, and prognosis were also examined.

Results: The frequency of CD4Foxp3 Tregs was comparable between HNSCC patients and age-matched benign tumor patients; however, CD45RAFoxp3 Tregs were significantly increased in HNSCC patients, in particular those with advanced stage tumors. The high frequency of CD45RAFoxp3 Tregs correlated with a poor prognosis and the low frequency of CD45RAFoxp3 Tregs before treatment showed a better clinical outcome, even in patients with advanced stage tumors. CD45RAFoxp3 Treg numbers were decreased after intensive treatments; however, Treg numbers recovered in the early stages of recurrent cases, even before the clinical manifestation.

Conclusion: CD45RAFoxp3 Tregs are associated with the clinical course of HNSCC and might be a new target for treatment and an early marker of tumor recurrence in HNSCC patients.
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http://dx.doi.org/10.1007/s00262-017-2021-zDOI Listing
October 2017

Invariant NKT cell-based immunotherapy for lung cancer and head and neck cancer.

Nihon Rinsho 2017 02;75(2):312-316

CD1d-restricted invariant natural killer T (iNKI) cells are the unique lymphocyte subpopu- lation that interacts with glycolipid via the invariant T cell receptor. Ligand activated iNKT cells produce a variety of cytokines, and thus bridging the innate and adaptive immune sys- tems. Since recent studies have highlighted iNKT cells to have potent anti-tumor effects, greater attention are being given to the development of iNKT cell-based immunotherapy for advanced solid tumors. This article summarizes the progress of our recent works on active immunotherapies aiming at the augmentation of iNKT cell function in vivo in patients with non-small cell lung cancer, and discusses the role of iNKT cell-induced immune responses in cancer immunotherapy.
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February 2017

Blockade of programmed death-1/programmed death ligand pathway enhances the antitumor immunity of human invariant natural killer T cells.

Cancer Immunol Immunother 2016 12 15;65(12):1477-1489. Epub 2016 Sep 15.

Department of Medical Immunology, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba, 260-8670, Japan.

The role of invariant natural killer T (iNKT) cells in antitumor immunity has been studied extensively, and clinical trials in patients with advanced cancer have revealed a prolonged survival in some cases. In recent years, humanized blocking antibodies against co-stimulatory molecules such as PD-1 have been developed. The enhancement of T cell function is reported to improve antitumor immunity, leading to positive clinical effects. However, there are limited data on the role of PD-1/programmed death ligand (PDL) molecules in human iNKT cells. In this study, we investigated the interaction between PD-1 on iNKT cells and PDL on antigen-presenting cells (APCs) in the context of iNKT cell stimulation. The blockade of PDL1 at the time of stimulation resulted in increased release of helper T cell (Th) 1 cytokines from iNKT cells, leading to the activation of NK cells. The direct antitumor function of iNKT cells was also enhanced after stimulation with anti-PDL1 antibody-treated APCs. According to these results, we conclude that the co-administration of anti-PDL1 antibody and alpha-galactosylceramide (αGalCer)-pulsed APCs enhances iNKT cell-mediated antitumor immunity.
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http://dx.doi.org/10.1007/s00262-016-1901-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5099366PMC
December 2016

Low Frequency of Programmed Death Ligand 1 Expression in Pediatric Cancers.

Pediatr Blood Cancer 2016 08 2;63(8):1461-4. Epub 2016 May 2.

Department of Medical Immunology, Graduate School of Medicine, Chiba University, Chiba, Japan.

Programmed death 1 (PD-1)/programmed death ligand 1 (PD-L1) pathway blockade has become a promising therapeutic target in adult cancers. We evaluated PD-L1 expression and tumor-infiltrating CD8(+) T cells in formalin-fixed, paraffin-embedded tumor specimens from 53 untreated pediatric patients with eight cancer types: neuroblastoma, extracranial malignant germ cell tumor, hepatoblastoma, germinoma, medulloblastoma, renal tumor, rhabdomyosarcoma, and atypical teratoid/rhabdoid tumor. One rhabdomyosarcoma with the shortest survival exhibited membranous PD-L1 expression and germinoma contained abundant tumor-infiltrating CD8(+) T cells and PD-L1-positive macrophages. The PD-1/PD-L1 pathway tended to be inactive in pediatric cancers.
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http://dx.doi.org/10.1002/pbc.26018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5074238PMC
August 2016

Akt1-mediated Gata3 phosphorylation controls the repression of IFNγ in memory-type Th2 cells.

Nat Commun 2016 Apr 7;7:11289. Epub 2016 Apr 7.

Department of Immunology, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan.

Th2 cells produce Th2 cytokines such as IL-4, IL-5 and IL-13, but repress Th1 cytokine IFNγ. Recent studies have revealed various distinct memory-type Th2 cell subsets, one of which produces a substantial amount of IFNγ in addition to Th2 cytokines, however it remains unclear precisely how these Th2 cells produce IFNγ. We herein show that phosphorylation of Gata3 at Ser308, Thr315 and Ser316 induces dissociation of a histone deacetylase Hdac2 from the Gata3/Chd4 repressive complex in Th2 cells. We also identify Akt1 as a Gata3-phosphorylating kinase, and the activation of Akt1 induces derepression of Tbx21 and Ifng expression in Th2 cells. Moreover, T-bet-dependent IFNγ expression in IFNγ-producing memory Th2 cells appears to be controlled by the phosphorylation status of Gata3 in human and murine systems. Thus, this study highlights the molecular basis for posttranslational modifications of Gata3 that control the regulation of IFNγ expression in memory Th2 cells.
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http://dx.doi.org/10.1038/ncomms11289DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4829694PMC
April 2016

Antibody-dependent cellular cytotoxicity toward neuroblastoma enhanced by activated invariant natural killer T cells.

Cancer Sci 2016 Mar 9;107(3):233-41. Epub 2016 Feb 9.

Department of Medical Immunology, Graduate School of Medicine, Chiba University, Chiba, Japan.

Anti-ganglioside GD2 antibodies mainly work through antibody-dependent cellular cytotoxicity (ADCC) and have demonstrated clinical benefit for children with neuroblastoma. However, high-risk neuroblastoma still has a high recurrence rate. For further improvement in patient outcomes, ways to maximize the cytotoxic effects of anti-GD2 therapies with minimal toxicity are required. Activated invariant natural killer T (iNKT) cells enhance both innate and type I acquired anti-tumor immunity by producing several kinds of cytokines. In this report, we investigated the feasibility of combination therapy using iNKT cells and an anti-GD2 antibody. Although some of the expanded iNKT cells expressed natural killer (NK) cell markers, including FcγR, iNKT cells were not directly associated with ADCC. When co-cultured with activated iNKT cells, granzyme A, granzyme B and interferon gamma (IFNγ) production from NK cells were upregulated, and the cytotoxicity of NK cells treated with anti-GD2 antibodies was increased. Not only cytokines produced by activated iNKT cells, but also NK-NKT cell contact or NK cell-dendritic cell contact contributed to the increase in NK cell cytotoxicity and further IFNγ production by iNKT cells and NK cells. In conclusion, iNKT cell-based immunotherapy could be an appropriate candidate for anti-GD2 antibody therapy for neuroblastoma.
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http://dx.doi.org/10.1111/cas.12882DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4814252PMC
March 2016

Invariant NKT cells are resistant to circulating CD15+ myeloid-derived suppressor cells in patients with head and neck cancer.

Cancer Sci 2016 Mar 13;107(3):207-16. Epub 2016 Feb 13.

Department of Otorhinolaryngology, Head and Neck Surgery, Graduate School of Medicine, Chiba University, Chiba, Japan.

Myeloid-derived suppressor cells (MDSC) are a heterogeneous population of immature and progenitor myeloid cells with an immunosuppressive role in various types of cancer, including head and neck squamous cell carcinoma (HNSCC). However, the effect on the host immune system, especially on invariant NKT (iNKT) cells with potent anti-tumor activity, remains unclear. In this study, we investigated the effects of circulating MDSC subsets on the peripheral lymphocytes of patients with head and neck tumors. A significant accumulation of CD15+ granulocytic MDSC (G-MDSC) and CD14+ monocytic MDSC (M-MDSC) was demonstrated in HNSCC patients. The percentage of G-MDSC showed an inverse correlation with the percentage of T cells in the peripheral blood. The increased G-MDSC was significantly associated with advanced clinical stage and poor prognosis of HNSCC patients. The proliferation and viability of T cells were suppressed by CD15+ cells, and the suppression was reversed by adding the hydrogen peroxide scavenger catalase. However, iNKT cell activation upon α-galactosylceramide (αGalCer) stimulation was not affected by the presence or absence of CD15+ G-MDSC. These results indicate that increased G-MDSC negatively affects peripheral T cell immunity, but not iNKT cells, in HNSCC patients, and that T cells are more sensitive to hydrogen peroxide produced by G-MDSC than iNKT cells. Cancer immunotherapy designed to enhance the antitumor activity of iNKT cells by stimulation with αGalCer may remain effective in the presence of G-MDSC.
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http://dx.doi.org/10.1111/cas.12866DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4814259PMC
March 2016